In chemotaxing cells, a polarity circuit involving PDKs and PTEN results in the accumulation of PI (3,4,5)P3 on the membrane at the cell's leading edge and serves to bias the local activity of the actin cytoskeleton. A similar circuit also plays a central role in cytokinesis. However, it is not known how signals are transmitted to PDKs and PTEN, how PI(3,4,5)P3 influences the local activity of the cytoskeleton, or which pathways act in parallel with the PI3K pathway. The answers to these questions have the potential to impact our understanding and treatment of cancer and inflammatory diseases. Dictyostelium discoideum is an excellent model system to pursue these investigations and has proved instrumental in identifying a number of "lead" genes that may be involved in these processes. The lead genes include YakA, a kinase that links receptor/G-protein to PI3K and other effectors;PH-domain containing proteins;Pianissimo (PiaA), a novel cytosolic regulator of chemotaxis that interacts with DdTor;RacC which may link PI(3,4,5)P3 to the cytoskeleton;a phospholipase A2 that synergizes with the PI3K pathway;Tsunami (TsuA) and Serrated (SerA), genes that control cell polarity. Cell lines that lack these and related genes have been generated and will be used to elucidate their function. Physiological responses such as PI(3,4,5)P3 accumulation, actin polymerization, and myosin phosphorylation will be quantified in these cells and the localization of components such as PI3K, PTEN, PI(3,4,5)P3, coronin, and myosin II will be determined by live cell imaging. To explore the relationship between these genes, cell lines lacking combinations of genes will be generated and, when genetic interactions are indicated by phenotype, further biochemical and imaging analyses will be pursued. Studies will also be conducted to try and discover additional genes involved in chemotaxis and cytokinesis. Proteins that interact physically with the known components will be identified using co-immunoprecipitation and yeast 2-hybrid analyses, and suppressor screens for mutations that reverse the chemotaxis and cytokinesis phenotypes of the existing cell lines will be used identify genes that have genetic interactions.